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lateral flow assay侧向层析检测资料

1 GE Healthcare
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From “Late Disease” To “Early Health”
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Earlier, More Targeted Treatment Track efficacy of treatment
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GE growth … our innovation
Global Research Center Headquarters Niskayuna, New York
Global Research— Europe Munich, Germany
John F. Welch Technology Centre Bangalore, India
China Technology Center Shanghai, China
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5 GE Healthcare
Diagnostics Products & Applications 诊断产品&应用
Flow Through Lateral Flow
Lateral Flow Assay
Flow Through Assay
Dipstick Assay
Sample Preparation
侧向层析法
Sample Wicking 样品吸收 Conjugate Release 结合物物释放 Membranes膜 Absorbents 吸收垫
渗滤法
? Membranes 膜 ? Absorbents 吸收垫
试纸检测法
? Cellulose Fiber 纤维素
样本制备
? Glass Fiber 玻璃纤维 ? Track Etched Membranes
径迹蚀刻膜
? Cast Membranes 浇注膜

Contents PART I 内容——第一部分
Diagnostics test systems Key parts of a lateral flow test Suggested range of products suitable for diagnostics Sample wicks Blood separation & other body fluids Conjugate release Absorbent & dip stick materials Why does quality matter? 诊断检测系统 侧向层析检测的主要部件 适用于诊断检测的产品 样品垫 全血及其他体液的分离 结合物释放 吸收垫及试纸条的材料 为什么质量很重要?
Diagnostics Test Systems 诊断 检测系统
Rapid diagnostics of pregnancy, drugs, cardiac markers, swine flue, HIV, etc. 怀孕、毒品、心肌标志物、猪流感、 艾滋病等的快速诊断。
Lateral Flow Assay
侧向层析检测
Sample Wicking 样品吸收 Conjugate Release 结合物释放 Membranes 膜 Absorbents 吸收垫
End Users and Professional Users 非专业及专业用户
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Diagnostics Test Systems 诊断 检测系统
Membrane-based immunochromatographic assay 基于膜的免疫层析检测
Lateral Flow Assay
侧向层析检测
Sample Wicking样品吸收 Conjugate Release 结合物释放 Membranes 膜 Absorbents 吸收垫
图1:典型的侧向层析检测条。 1=样品垫,2=结合物释放垫, 3=检测线,4=反应膜,5=对照 线,6=吸收垫,7=背衬。
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Lateral Flow Test 侧向层析检测
Particle Conjugate 结合物 Sample Pad 样品垫 Control Line 对照线 Wick 吸收垫
Test Line 检测线
Nitrocellulose Membrane 硝酸纤维素膜
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Diagnostics Principle 诊断 原理
图2: 由于夹心法侧向层析测试需要检测物至少提供两 个抗原表位,因此通常只用于大分子待测物的检测。阳 性检测会显示两条线,而阴性检测只出现一条线(对照 线)。竞争法检测用于不具备有两个独立的抗体结合位 点的小分子待测物的检测。在这种检测中,用于捕获待 测物的抗体被固定化的竞争剂(待检测物质的类似物) 取代。当样品中存在待检测分子,识别抗体的结合位点 被这些分子占据,这使得结合物与检测线的结合不复存 在(如待分析物质和竞争剂争夺吸附结合物)。 1=大分子待检测物,2=结合物,3=捕获抗体(根据分 析物情况而定),4=对照线抗体(如anti-IgG),5=小 分子待测物,6=竞争剂。
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Diagnostics Test Systems 诊断 检测系统
Rapid diagnostics (3 to 5 min) of e.g. antibodies (serology), etc. Professional users
Flow Through Assay
渗滤检测
Membranes 膜 Absorbents 吸收垫
抗体(血清学)等的快速检测 (3到5分钟)。 适合专业用户使用
图23:典型的渗滤法装置的组成。 1=检测盒。2=反应膜。3=吸收垫。 4=封闭试剂。
Professional Users 专业用户
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Diagnostics Test Systems 诊断 检测系统
Membrane strips with antigen or antibody lines to detect autoimmun diseases etc. Often used as “homebrew”test made by diagnostic labs 带有抗原线或抗体线的膜条可 用于检测自身免疫性疾病等。 通常由诊断实验室自制使用。
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Line Assay 谱线分析 Western Blots 蛋白印迹
Membranes 膜
Professional Users专业用户
Diagnostics Test Systems 诊断 检测系统
Easy “dip-into body-fluid” – “colour-reaction” tests. E.g. Urine dip-sticks (Roche)
Dipstick Assay
试纸检测
Cellulose Fiber
简易的试纸-比色法检测 如:尿试纸(罗氏) Professional Users专业用户
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纤维素

Diagnostics Test System 诊断 检测系统
Flow Through Lateral Flow
Sample prep by filtration. Capturing of cells on membrane surface (TEM), removal of blood cells, etc.
Sample Preparation
样品制备
Glass Fiber 玻璃纤维 TEM 径迹蚀刻膜 Cast Membranes 浇注膜
通过渗流制备样品 在膜表面捕获细胞(径迹蚀刻 膜)、去除血细胞等。
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Contents PART I内容——第一部分 Membranes in lateral flow tests Key Properties Suggested range of products Why does quality matter? 侧向层析检测中的膜 主要特性 产品的归类 为什么质量很重要?

Key Parts of a Rapid Test (Focus: Lateral Flow Test) 快速检测的重要成员(重点关注:侧向层析检测) Sample Preparation Conjugate Release Membranes Absorbents PART II 第二部分 Nitrocellulose Membranes as the reaction part of a test 作为检测中反应部件的硝酸纤维素膜 样品制备 结合物释放 膜 吸收垫
Membranes in Lateral Flow Assays – Key Properties 侧向层析检测中的膜——主要特性
The membrane is a matrix which carries and preserves at least two different capture reagents It must allow the mixing of materials and their interaction with the capture lines It must allow fluid flow 至少结合两种不同的捕获试剂 必须在捕获线上能使材料和与之作用的物质混合 必须能使液体流过
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Membranes in Lateral Flow Assays- Structure 侧向层析检测中的膜——结构 Membranes are being made by a random precipitation of NC fibres from a solution Best comparison: A sponge 膜由溶液中的硝酸纤维随机沉淀而来 最好的比喻:海绵
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Structure of Nitrocellulose 硝酸纤维的结构
Scanning Electron Micrograph of a Whatman AE 100 membrane. Nominal pore size 12 μm. Whatman AE 100膜的电镜扫描图。标称孔径12 μm。
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Air Side and Belt Side of Nitrocellulose Membranes 硝酸纤维素膜的气面和带面
Whatman AE 100膜在500倍放大倍数下的SEM图像。 左图:空气面。右图:皮带面。 这种非对称性在大孔径膜上表现的更加明显,如AE100,其标称孔径为12μm。
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Characterisation of Membranes 膜的特性 Two parameters are used in the literature:
? Pore size ? Capillary rise time
文献中使用的两个参数:
? 孔径 ? 毛细上升时间
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Distance travelled against time 随时间迁移的距离
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Distance travelled against time 随时间迁移的距离
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Problems with pore size definition 孔径精确度的问题
5 Measured pore size (microns) 4 3 2 1 0 0 5 10 15 Nominal pore size (microns)
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Correlation between nominal pore size and capillary rise time for Whatman membranes Whatman膜的标称孔径及毛细上升时间之间的相关性
Nominal pore size (μm)
3 5 8 12
Time to wick 4 cm (sec)
245 185 140 100
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Change of Capillary Rise Rate Depending on Migration Distance 毛细上升率随迁移距离的改变情况
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Effect of wicking rate on apparent analyte concentration 浸润率对被测物表观浓度的作用
Distance of Lateral wicking rate Apparent capture line from (cm/15secs) concentration of origin (cm) analyte 2 1.9 1 2.5 0.7 7.4 3 0.3 40
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Assay sensitivity decreases with increasing capillary flow rate 检测灵敏度随着毛细上升率的升高而降低
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Level of binding observed on different membranes for IgG 不同膜对IgG的结合水平
200 Relative Intensity 180 160 140 120 100 3 5 8 12 Membrane pore size (micron)
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Type A Type B Type C

Level of binding observed on different membranes for BSA 不同膜对BSA的结合水平
180 Relative Intensity 160 140 120 100 3 5 8 12
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Type A Type B Type C
Membrane pore size (micron)
Why do proteins bind to nitrocellulose? 蛋白为什么能够与硝酸纤维结合?
What forces are acting? ? Electrostatic interaction ? Hydrophobic interaction What is the effect of each attractive force? ? Partition of protein from solution ? Long term interaction 什么作用力在起作用? ? 静电作用 ? 疏水作用 每种力量所起的特别作用分别是什么? ? 令蛋白从溶液中分离 ? 长时间的作用力
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The adsorption process 吸附过程
Protein in bulk solution 原液中的蛋白质 Transport to interfacial region 迁移到界面区 Adsorption 吸附 Rearrangement on surface 在表面上重新排列
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Protein binding to a solid phase 蛋白的固相结合
The binding of protein to a solid phase is a multi-stage process It is dependent upon: ? Diffusion to the surface ? Separation of the protein from the solvent to the solid phase ? Adsorption to the surface ? Rearrangement to the lowest energy state 蛋白的固相结合是一个多步过程 依靠的是: ? 表面扩散 ? 将蛋白从溶液分离到固相上 ? 表面吸附 ? 重新排列为低能量状态
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Multipoint addition 多点附着
The addition is reversible, when there is only 1 binding site the protein will desorb from the surface 附着过程是可逆的,当只有一个结合点时蛋白会随时从表面释放
The binding at each individual point is reversible, however with multiple points of attachment the protein remains bound 每个点的附着是可逆的,然而随着多个附着点的存在,蛋白将维 Proprietary internal information for GE internal training purposes only! 持结合状态
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General principles 常规原理
The importance of protein binding Factors that influence protein binding ? The application buffer ? The membrane used ? The protein (capture reagent) itself ? The application system and ambient conditions 蛋白结合的重要性 影响蛋白结合的因素 ? 反应缓冲液 ? 反应膜 ? 蛋白(捕获剂)本身 ? 检测系统及反应条件
The aim of a lateral flow test is to achieve a clear and crisp test line. This largely depends on correct binding of the capture reagent to the NC membrane 侧向层析检测的目的是获 得一条清晰分明的检测线 。这极大依赖于捕获试剂 与NC膜的准确结合。
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The application buffer 使用的缓冲液
No universal buffer, each system must be optimised Golden rule: “to maximise protein binding, the protein should be as unstable as possible in solution” Challenge: You must also preserve as many epitopes as possible! 不存在通用缓冲液,每一个检测系统都必须经过优化 金标准: “使溶液中的蛋白尽可能的不稳定,从而使蛋白得到最大结合” 挑战:你需要保留尽可能多的抗原决定簇!
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Optimisation of application buffer 优化反应缓冲液
Ionic strength ? Important for partitioning pH ? At its’ pI a protein is least stable in solution Co-precipitating agent ? Usually an alcohol to make protein unstable Buffer salt ? Does not necessarily matter, but: ? For some epitopes, the presence of specific ions may be vital ? For some proteins a zwitterionic salt helps with binding 离子强度 ? 对蛋白在溶液中的分配很重要 酸碱度 ? 溶液中的蛋白在其等电点时最不稳定 共沉淀剂 ? 通常乙醇能使蛋白不稳定 盐溶液 ? 不是必要因素,然而: ? 对某些抗原决定簇,特定离子的存在 可能是必需的 ? 两性离子盐对某些蛋白的结合有所帮 助
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Recommended starting buffer 推荐的起始溶液 10mM Phosphate pH 7.0 A low concentration of NaCl 3% Ethanol
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Varied results from capture lines of 1 mg/ml mouse IgG applied using different buffers: (a) 10 mmol phosphate, pH 7.2; (b) 10 mmol phosphate, + 3% methanol, pH 7.2; (c) 10 mmol phosphate + 150 mmol NaCl + 3% methanol, pH 7.2; (d) 50 mmol phosphate + 150 mmol NaCl + 1% BSA, pH 7.2; (e) 50 mmol phosphate + 150 mmol NaCl, pH 7.2; (f) 50 mmol phosphate + 150 mmol NaClpH 6.0. All samples were detected by a 40 nmol gold-conjugated goat antimouse IgG antibody. 不同缓冲液中1mg/ml的鼠IgG样品所获得的不同的捕获线结果: (a) 10 mmol 磷酸盐, pH 7.2; (b) 10 mmol 磷酸盐, + 3% 甲醇, pH 7.2; (c) 10 mmol 磷酸盐 + 150 mmol NaCl + 3% 甲醇, pH 7.2; (d) 50 mmol 磷酸盐 + 150 mmol NaCl + 1% BSA, pH 7.2; (e) 50 mmol 磷 酸盐 + 150 mmol NaCl, pH 7.2; (f) 50 mmol 磷酸盐 + 150 mmol NaCl, pH 6.0. 所有样品 皆使用40nmol胶体金偶联的羊抗鼠IgG抗体进行测试。
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